Mauro Donaldo Saucedo-Plascencia I know two strains of plant pathogenic bacteria that lost original pathogenic phenotype (changed symptoms produced on plants) at the same time in 3 different places. They were stored at -80 or in sterile water at +20oC. One of these strains stored lyophilized kept original phenotype. The fact of such change was published but without information about simultaneous change.

have stored thousands of pathgenic E coli at -80C, and never encountered this.

it already happened to me but not when they were stored at -80 C but when they are handled a lot and kept in the fridge.

It's possible if your strain has been subcultured for many times. If the sample is taken from the patient(for example) and then store at -80, it's fine. But if your strain comes from another lab, then it's quite possible for the bacteria to lose their virulence. Culturing bacteria in lab condition cannot provide enough environmental stress for keeping virulence sometime.

I have used strains frozen in the 70s that still maintain there virulence. However culturing and especially manipulation eg making competent and/or electroporation can result in loss of virulence plasmids. It also depends on how your testing this. Certain conditions would need to be met to switch on particular virulence factors. For example subculturing in DMEM for T3SS.

I just want to thank you for your time and attention to my question, I really appreciate it Mitchell Pallett Leticia Matter Richard Goldstein Alex Ignatov

Further. In addition to clinical pathogenic isolates of e. coli, we have also stored H. influenza, P. aerugionosa, B. cepacia and S. pneumonia clinical isolates at -80 C mixed with glycerol. Never detected loss of virulence genes.

YES, Virulence gene expression in most bacteria is a highly regulated phenomenon, affected by a variety of parameters including osmolarity, pH, ion concentration, iron levels, growth phase, and population density. Virulence genes are also regulated by temperature, which acts as an `on-off' switch in a manner distinct from the more general heat-shock response. Here, we review temperature-responsive expression of virulence genes in four diverse pathogens....[https://www.sciencedirect.com/science/article/abs/pii/S1286457900002720]

Yes, this happens some times if the virulence genes are located in a plasmid. Bacteria may loose that plasmid and as a result the virulence is lost.

Yes, this happens some times but before using that preserved isolates go for lab animal inoculation and reisolate the organism.

Yes, it is possible for pathogenic bacteria to lose their virulence genes after freeze storage, particularly if the bacterial strain has been subcultured multiple times under laboratory conditions. Freeze storage itself does not typically cause the loss of virulence genes, but the subculturing process and adaptation to laboratory conditions can result in genetic changes that may impact virulence.

When bacteria are repeatedly subcultured in the laboratory, they can undergo genetic changes, including the loss or mutation of certain genes. This can occur due to genetic drift, selective pressures, or the absence of specific environmental cues that are present in their natural host or infection site. Over time, these genetic changes can lead to the loss or alteration of virulence factors, rendering the bacteria less pathogenic.

Additionally, laboratory culturing conditions may not fully replicate the complex and dynamic environments that bacteria encounter in their natural hosts or infection sites. This can result in the downregulation or loss of virulence genes that are not essential for survival and growth under laboratory conditions.

It is important to note that the extent of virulence gene loss or alteration can vary depending on the specific bacterial strain, the virulence factors involved, and the conditions of freeze storage and subculturing. Therefore, when working with pathogenic bacteria such as diarrheagenic E. coli, it is recommended to carefully monitor and characterize the strain throughout the research process, including periodic assessments of virulence gene expression or phenotypic virulence assays, to ensure the maintenance of the desired virulence properties.